Abstract

Objective: The objective of this thesis is to elucidate the differences between hi glucose and normal human cutaneous microvascular endothelial cells (HMEC-1) to gain more insight into which metabolic and motility mechanisms are impaired in diabetes. In this thesis, we will also assess the pro-angiogenic properties of Eu(OH)₃ nanorods using an in vivo and in vitro diabetic model. Methods: The differences between hi glucose and normal glucose HMEC-1 cells were determined by proliferation assays, wounding assays, IMF for cytoskeletal associated proteins- VE-Cadherin and F-Actin, and RT-PCR. The pro-angiogenic properties of Eu(OH)₃ nanorods were determined using a quantitative angiogenesis assay using the chorioallantoic membrane (CAM) and a diabetic mouse cutaneous wound healing model. Results: We observed that normal glucose (5.56 mM) HMEC-1 cells are more proliferative than cells in hi glucose (25 mM). Normal HMEC-1 cells wound decreased to 2.42 units and that of diabetic cells was 4.59 units. More cell migration into the wound site is seen with normal endothelial cells. Normal HMEC-1 cells have more F-actin at the leading wound edge than hi glucose HMEC-1 cells and at the wound edge, normal cells are observed to have F-actin filaments that are directed to the wound site rather than poorly directional as seen with hi glucose cells. Normal HMEC-1 cells showed more VE-Cadherin junctions compared to hi glucose cells of the same density. Hi glucose HMEC-1 cells treated with 50 ng/mL VEGF for 8, 24, and 48 hours glucose HMEC-1 cells did not show any significant change in expression of VE-Cadherin compared to no treatment. We observed similar number of branch points between VEFG Vascular Endothelial Growth Factor) and 20μg of Eu(OH)₃. There was a significant increase in branch points when changing the amount of Eu(OH)₃ from 1μg to 20μg, independent of medium. When compared to VEGF induced angiogenesis, Eu OH)3 nanorods appeared to form a more complicated vascular network due to the variety in the sizes of the vessels being formed. In diabetic mice we found that cutaneous wound area decreased faster for mice treated with Eu(OH)₃ nanorods than those treated with control vehicle [62% (Europium) Vs 43% (Control) wound area reduction at Day 18]. Additionally, the mice treated with Eu(OH)₃ showed a relatively sustained thermal profile with minimal flare, a surrogate marker of inflammation, although this was not statistically significant. This suggests. Conclusion: Hi glucose cutaneous human microvascular endothelial cells (HMEC-1) show impaired proliferation, delayed migration into a wound site, and decreased VE-cadherin and F-actin compared to normal. These impairments must be addressed when developing a treatment to improve angiogenesis and wound healing in diabetic patients. Eu(OH)₃ nanorods are bioactive nanoparticles and induce angiogenesis which results in relatively faster skin wound healing in diabetics. Subsequent studies will direct inquiry into the mechanism of the nanoparticle through analysis of gene expression of candidate genes, such as VEGF.

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